[74] Electron Spin Resonance Oximetry

Nitroxides, the relatively stable free radicals, are used as spin probes in electron spin resonance (ESR) spectroscopy for a variety of chemical and biological purposes. They have been shown, for instance, to be efficient tools for the determination of cell or organelle volumes, as well as of bioenergetic parameters such as pH and potential gradients. As molecular oxygen is paramagnetic, measurements of line width should lead to its quantitation. The chapter describes this methodology, recently termed ESR oximetry, as applied to total and intracellular measurements of dissolved O₂ in a cyanobacteria system. The organism used in the work described here is the unicellular, marine species Agmenellum quadruplicatum. Two main approaches have been developed in ESR oximetry. In the first, it is not the width of the main line itself which is being followed, but rather the height of the superhyperfine lines, which tend to “flatten out” on signal broadening. This phenomenon, and several variations, has been applied to O₂ measurements with a high degree of accuracy and sensitivity. Oxygen-induced line broadening should theoretically be independent of probe concentration. At high probe densities, however, self-quenching, owing to interactions among the probe molecules, may lead to an increase in line width.